Engineered Branaplam Aptamers Exploit Structural Elements from Natural Riboswitches

Drug candidates that fail in clinical trials for efficacy reasons might still have favorable safety and bioavailability characteristics that could be exploited. A failed drug candidate could be repurposed if a receptor, such as an aptamer, were created that binds the compound with high specificity. Branaplam is a small molecule that was previously in development to treat spinal muscular atrophy and Huntington’s disease. Here, we report the development of a small (48-nucleotide) RNA aptamer for branaplam with a dissociation constant of ∼150 nM. Starting with a combinatorial RNA pool integrating the secondary and tertiary structural scaffold of a Guanine-I riboswitch aptamer interspersed with regions of random sequence, in vitro selection yielded aptamer candidates for branaplam. Reselection and rational design were employed to improve binding of a representative branaplam aptamer candidate. A resulting variant retains the pseudoknot and two of the paired elements (P2 and P3) from the scaffold but lacks the enclosing paired element (P1) that is essential for the function of the natural Guanine-I riboswitch aptamer. A second combinatorial RNA pool based on the scaffold for TPP (thiamin pyrophosphate) riboswitches also yielded a candidate offering additional opportunities for branaplam aptamer development.


Enzymes
Taq DNA polymerase and T4 polynucleotide kinase were obtained from New England Biolabs.SuperScript III and TURBO DNase were obtained from Invitrogen.rAPid alkaline phosphatase was obtained from Roche.RNase T1 was obtained from Thermo Scientific.All enzymes were used with the provided buffers and recommended reaction conditions following the manufacturer's instructions, unless stated otherwise.T7 RNA polymerase was purified in-house.

Synthesis of S7-S12 G0 Pool
For each of the six scaffolds S7-S12, the corresponding forward and reverse oligonucleotides (100 pmol each) for each scaffold were mixed as shown below: To each of the six solutions, 5 µL 10 mM dNTPs and deionized, sterile H2O (dH2O) were added to a final volume of 37.5 µL.Each solution was heated to 90 °C for 1 min, and subsequently allowed to cool to room temperature on the benchtop (~3 min).Then, 10 µL 5x First Strand buffer (provided by the manufacturer), 5 µL 0.1 M DTT, and 2 µL SuperScript III RT were added to each solution.Primer extension was performed by incubating each solution at 55 °C for 1 h.The RT was heat-denatured by incubating at 75 °C for 15 min, but no further purification was performed.
12.5 µL (~25 pmol DNA) from each of these reactions was used in subsequent in vitro transcription reactions, which also contained 55 µL dH2O, 10 µL 10x transcription buffer [150 mM MgCl2, 20 mM spermidine, 500 mM Tris (pH 7.5 at ~20 °C), and 50 mM DTT], 20 µL 10 mM NTPs, and 2.5 µL T7 RNA polymerase.Each reaction was incubated at 37 °C for 2 h.To deplete template DNA, 1 µL Turbo DNase was added and each reaction was incubated at 37 °C for 10 min.The resulting RNA pool was purified by denaturing (8 M urea) 10% polyacrylamide gel electrophoresis.The band containing the target RNA was excised from the gel, excluding the top and bottom 20% of the band.RNA was eluted from the gel by crushing and soaking in buffer [0.4 M NaCl, 10 mM Tris, 1 mM EDTA (pH 8 at ~20 °C)] and subsequently concentrated by precipitation in ethanol.

In vitro Selection (Scaffolds S7 through S12)
In vitro selection was performed as previously described. 1Briefly, the G0 RNA pool was mixed with a 10x molar excess of capture oligonucleotide in selection buffer (20 mM HEPES, 100 mM KCl, 3 mM NaCl (total [Na + ]: 10 mM), and 1 mM MgCl2, pH 7.5 at ~20 °C) and the resulting solution was incubated at 90 °C for 1 min.The RNA-capture oligonucleotide solution was loaded onto a Micro-Bio spin column loaded with streptavidin-agarose.After stringent washing, a solution containing 10 µM branaplam (as well as three other target compounds) was loaded onto the column.RNA molecules that eluted in the presence of the target compounds were amplified by reverse-transcription polymerase chain reaction (RT-PCR).This process was repeated 11 times, resulting in the G11 population.During selection rounds 1 through 6, the column was incubated with the target ligand solution for 2.5 min.During selection rounds 7 through 11, the incubation time was shortened to 0.5 min.

In vitro Selection (TPP Scaffold)
The process was performed as described above for scaffolds S7 through S12. 14 rounds of in vitro selection were performed, resulting in the G14 population.

Branaplam Reselection
The branaplam reselection (BRS) template was designed based on the sequence of BRS-G11-R1B with different primer-binding regions.75 nucleotides between the primer-binding regions were mutagenized at 6% degeneracy.The selection was performed as described above, except that the target solution contained 100 nM branaplam.Ten selection cycles were performed, resulting in the BRS-G10 population.

Elution Profiling
Elution profiles were performed as previously described. 1Briefly, RNA was synthesized by in vitro transcription with T7 RNA polymerase, desphosphorylated with alkaline phosphatase, and radiolabeled with T4 polynucleotide kinase in the presence of γ-32 P-ATP.Elution profiles were performed similarly to the selection.1 µL of each solution was pipetted onto filter paper and phosphor imaged using a Typhoon scanner.

In-line Probing
In-line probing was performed as previously described. 2Briefly, 5′-32 P-labeled RNA was mixed with the ligand at the specified concentration and 2x in-line probing buffer (20 mM MgCl2, 100 mM KCl, 50 mM Tris-HCl [pH 8.3 at ~20 °C]).After incubating at room temperature for ~48 h, the reaction was quenched by the addition of 2x loading buffer [18 M urea, 20% w/v sucrose, 0.1% w/v sodium dodecyl sulfate, 0.05% w/v bromophenol blue, 0.05% xylene cyanol, 90 mM Tris, 90 mM borate, 1 mM EDTA pH 8.0 at ~20 °C)].RNase T1, which cleaves at every G nucleotide, was used according to the manufacturer's instructions to generate a "T1 ladder" for each RNA.
-OH ladders were prepared by treatment with 50 mM sodium bicarbonate at 90 °C for 1 min.The 10 samples were analyzed by 10% denaturing (8 M urea) PAGE.The resulting gels were dried and subsequently phosphor imaged using a Typhoon scanner.Densitometry was performed with ImageJ software.

Next-generation Sequencing
50 ng of the corresponding DNA population were submitted to the Yale Center for Genomic Analysis.The populations were sequenced at a depth of about 40 million reads.Paired-end reads were sequenced with a read length of 150 base pairs.

Bioinformatics
Computational sequence analysis was performed as described previously. 1 The python script toTally was used to calculate percent abundances of unique sequences in each sequenced population. 3The python script selfishCluster was used to generate clusters of similar sequences. 3finder was used to generate Stockholm files (.sto) containing putative structural information. 4R was used to draw the output of CMfinder. 5fraction of RNA bound.The KD for this interaction is 2.44 ± 1.03 µM.The KD for this interaction is 1.17 ± 0.17 µM.

Figure S3. Branaplam reselection pool
RNA pool design for branaplam reselection.The region highlighted in green is mutagenized at 6% degeneracy.The flanking regions highlighted in grey are the primer-binding regions, which were not mutagenized.

Figure S4. Elution profile of the G10 reselection population
Autoradiogram of an elution profile with radiolabeled G10 reselection population depicting the relative amounts of radioactivity from sequential elutions.Key -UR: unbound RNA, Wn: wash with selection buffer in which n corresponds to the number of washes that have been performed, Bn: elution with 100 nM branaplam dissolved in selection buffer in which n corresponds to the number of elutions that have been performed.the fraction of RNA bound.The KD for this interaction is 2.27 ± 0.15 µM.
Figure S1.In-line probing of aptamer 11-1 with branaplam A. Secondary structure diagram of 11-1, which comprised 13.1% of the G11 population in the original selection.B. Autoradiogram of a polyacrylamide gel showing the result of in-line probing reactions containing radiolabeled 11-1 RNA and increasing branaplam concentrations ranging from 10 -9 to 10 -5 M at quarter-log intervals.C. Plot of the logarithm of branaplam concentration vs.

Figure S2 .
Figure S2.In-line probing of truncated aptamer 11-1B with branaplam A. Sequence and secondary structure model of 11-1B.B. Autoradiogram of a polyacrylamide gel showing the result of in-line probing reactions containing radiolabeled 11-1B RNA and increasing branaplam concentrations ranging from 10 -9 to 10 -5 M at quarter-log intervals.C. Plot of the logarithm of branaplam concentration vs. the fraction of RNA bound.The KD for this interaction

Figure S5 .
Figure S5.In-line probing of r10-1 with branaplam A. Sequence and secondary structure model of r10-1 RNA, which comprised 18.4% of the G10 reselection population.B. Autoradiogram of in-line probing gel with 5′-32 P-labeled r10-1 RNA incubated with increasing branaplam concentrations ranging from 10 -9 to 10 -5 M at quarter-log intervals.C. Plot of the logarithm of branaplam concentration vs. the fraction of RNA bound.The KD for this interaction is 420 ± 41 nM.

Figure S6 .
Figure S6.In-line probing of r10-2 with branaplam A. Sequence and secondary structure model of r10-2 RNA, which comprised 5.2% of the G10 reselection population.B. Autoradiogram of in-line probing gel with 5′-32 P-labeled r10-2 RNA incubated with increasing branaplam concentrations ranging from 10 -9 to 10 -5 M at quarter-log intervals.C. Plot of the logarithm of branaplam concentration vs. the fraction of RNA bound.The KD for this interaction is 623 ± 55 nM.

Figure S8 .
Figure S8.In-line probing of r10-4 with branaplam A. Sequence and secondary structure model of r10-4 RNA, which comprised 4.1% of the G10 reselection population.B. Autoradiogram of in-line probing gel with 5′-32 P-labeled r10-4 RNA incubated with increasing branaplam concentrations ranging from 10 -9 to 10 -5 M at quarter-log intervals.C. Plot of the logarithm of branaplam concentration vs. the fraction of RNA bound.The KD for this interaction is 634 ± 74 nM.

Figure S9 .
Figure S9.In-line probing of r10-1C and r10-1C-M1 with branaplam A. Sequence and secondary structure model of r10-1C RNA.The nucleotides highlighted in green indicate the location of a putative pseudoknot.The r10-1C-M1 variant contains two G to A mutations at nucleotides 36 and 37 as shown.B. Autoradiogram of in-line probing gel with 5′-32 Plabeled r10-1C RNA incubated with increasing branaplam concentrations ranging from 10 -9 to 10 -5 M at quarter-log intervals.C. Autoradiogram of in-line probing gel with 5′-32 P-labeled r10-1C-M1 RNA incubated with increasing branaplam concentrations ranging from 10 -9 to 10 -5 M at quarter-log intervals.D. Plot of the logarithm of branaplam concentration vs. the fraction of r10-1C RNA bound.The KD for this interaction is 400 ± 43 nM.

Figure S11 .
Figure S11.In-line probing of r10-1H with branaplam A. Sequence and secondary structure model of r10-1H RNA.B. Autoradiogram of in-line probing gel with 5′-32 P-labeled r10-1H RNA incubated with increasing branaplam concentrations ranging from 10 -9 to 10 -5 M at quarter-log intervals.C. Plot of the logarithm of branaplam concentration vs. the fraction of RNA bound.The KD for this interaction is 311 ± 19 nM.

Figure S16 .Figure S17 .
Figure S16.In-line probing of r10-1Q with branaplam A. Sequence and secondary structure model of r10-1Q RNA.B. Autoradiogram of in-line probing gel with the indicated 5′-32 P-labeled RNAs incubated with increasing branaplam concentrations ranging from 10 -5 to 10 -9 M at quarter-log intervals.

Figure S21 .
Figure S21.In-line probing of t14-4 with branaplam A. Sequence and secondary structure model of t14-4 RNA.B. Autoradiogram of in-line probing gel with 5′-32 P-labeled t14-4 RNA incubated with increasing branaplam concentrations ranging from 10 -8 to 10 -4.67 M at third-log intervals.C. Plot of the logarithm of branaplam concentration vs.